MAPK). Attenuation of the MAPK-inflammatory/apoptotic pathway in SH-SY5Y cells concurrent with protection of rotenone-triggered motor impairment in rats, is a manifestation of the combined antioxidant/anti-inflammatory activity of SD and SDA together with levodopa release. The concept of joined therapies into a single molecule, where levodopa precursors confer antioxidant activity by enabling NAC delivery across the BBB, provides a potential disease-modifying treatment for slowing PD progression.Parkinson’s disease (PD) is characterized by a gradual degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpC). Levodopa, the standard PD treatment, provides the missing dopamine in SNpC, but ultimately after a honeymoon with levodopa treatment the neurodegenerative process and the progression of the disease continue. Aimed at prolonging the life of dopaminergic cells, we prepared the levodopa precursors SuperDopa (SD) and SueprDopamide (SDA), in which levodopa is merged with the antioxidant N-acetylcysteine (NAC) into a single molecule. Rotenone is a mitochondrial complex inhibitor often used as experimental model of PD. In vivo, SD and SDA treatment show a significant relief of motor disabilities in rotenone-injected rats. SD and SDA also lower rotenone-induced-α-synuclein (α-syn) expression in human SH-SY5Y cells, and α-syn oligomerization in α-syn-overexpressing-HEK293 cells. In the neuronal SH-SY5Y cells, SD and SDA reverse oxidative stress-induced phosphorylation of cJun-N-terminal kinase (JNK) and p38-mitogen-activated kinase (p38 Neurodegenerative diseases share neuroinflammation as a common mechanism resulting from activated microglia that release pro-inflammatory cytokines. The loss of 9-type dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) has been shown to be responsible for the first motor symptoms of Parkinson’s disease (PD).Virtually all PD patients will require levodopa therapy. However, PD progresses with time, and patients experience diminished duration of benefit from each dose. The diminished effectiveness over time is also accompanied by the development of medication-related complications such as motor fluctuations, and levodopa-induced dyskinesia . The decMAPK)  during the oxidative metabolism of dopamine within the DA cells, and impaired bioenergetics in the mitochondria contribute to premature cell death. Oxidative stress also triggers the mitogen-activated protein kinases (MAPKs) inflammatory/apoptotic pathways through the phosphorylation of cJun N-terminal kinase (JNK) and p38-mitogen protein kinase (p38 review –7)..MAPK) (rA53T) A9 DA neurons [Mitochondrial toxins including herbicide paraquat, pesticide rotenone, or the funcocide MB-manganese, target a variety of redox-sensitive proteins in the brain contributing to sporadic PD . These t neurons .A53T are linked to genetic mutations that generate intracellular inclusions called Lewy body that contribute to the neuronal dysfunction and pathology of DA cells [Oxidative/nitrosative stress induced by paraquat has been shown to modify α-syn by nitration of a tyrosine residue and oxidation of a methionine residue, thereby contributing to its aggregation –13. ThesDA cells –17. ThesDA cells –20.To develop a disease-modifying strategy for PD, we designed two dopamine precursors, AcCys-L-Dopa-Cys-amide, called SuperDopa (SD), and AcCys-L-Dopa amide, called SuperDopAmide (SDA).SD is a family member of the thioredoxin mimetic peptides (TXM-peptides). TXM- peptides protect neuronal and non-neuronal cells from apoptosis in vitro and in vivo, by inhibiting the oxidative stress-induced MAPK-inflammatory/apoptotic pathway and catalyzing S-denitrosylation , 21–30.SDA is a dipeptide comprising of N-acetylcysteine (NAC) and DopAmide. DopAmide itself is a levodopa precursor that confers a sustained release of dopamine in 6-OH-dopamine-lesioned rats .To recapitulate in vivo features of SD and SDA we used the highly reproducible rotenone rat model, in which rotenone-treated rats develop PD features like bradykinesia, postural instability and/or rigidity, thus providing an excellent tool to test potential PD neuroprotective reagents . In vitrAs demonstrated, both SD and SDA appeared to combine anti-inflammatory/antioxidant activities with the ability to replenish the cells with levodopa. The impact of joined activities into a single molecule is often greater than administrating each molecule separately.The concept of combined therapies of levodopa precursor with antioxidant activity into a single molecule that enables NAC delivery across the BBB and inhibition of the neurodegenerative process, could become a disease-modifying treatment with an outlook for slowing PD progression.SD (AcCys-L-DOPA-Cys-amide) is N-acetylated tri-peptide comprising of two Cys residues that flank levodopa Fig. ; upper. SDA (N-Acetylcysteine-L-DOPA-amide) is an N-acetyl blocked dipeptide comprising of a single Cys and a single residue of levodopa modified at the carboxy-terminal to an amide. SDA was synthesized , and its purity (98%) and molecular weight were determined by HPLC Fig. S, and masHere, SD and SDA were tested in vivo using the rotenone rat model, which mimics PD motor dysregulations and is one of the most reliable animal models of PD. Three groups of rats were injected intraperitoneally (i.p) in the morning with rotenone (3.0 mg/kg/day) in a specialized vehicle, for 9 days. In the afternoon, one group was i.p injected with SDA (33 mg/kg/day) and another group, with SD (33 mg/kg/day), for 9 days (“Methods”).Body weight was monitored on days 4, 7, and 10 Fig. . A slighMotor coordination and balance were evaluated using the rotarod behavior test at days 4, 7, and 10 Fig.  left. ThThe rearing cylinder test was used to evaluate locomotor activity in rodent models of CNS disorders. A gradual decrease in the ability of the rotenone-treated rats to place the palm on the wall of the cylinder was monitored at days 4, 7, and 10, indicating impaired body support Fig. , left. RRat beam walk test was applied for examining SDA and SD effects on motor coordination and balance in the rotenone rat model. The goal of this assay was to test the rat ability to stay upright and walk across an elevated narrow beam to a safe platform. After 2 days training, the assay examined the time it takes for the rat to traverse the beam and the number of paw slips that occur in the process. This test is complementary to the rotarod, and can detect subtle deficits in motor skills and balance.A significant improvement in the time required for terminating the walk was observed in the rotenone/SD or rotenone/SDA treated rats, compared to rotenone/only, at days 4, 7, and 10 Fig.  left. QuTo assess a putative neuroprotective mechanism, and cell permeation we examined the antioxidant/anti-inflammatory activities of SD and SDA, by monitoring the oxidative stress-induced MAPK-apoptotic/inflammatory pathway in human neuroblastoma SH-SY5Y cells. Oxidative stress was induced by auranofin (Auf), an inhibitor of thioredoxin reductase. SH-SY5Y cells are often used as a cellular model of various neurological oxidative stress-related disorders such as PD and Alzheimer’s disease , 34.Initially, we explored the effects of Auf on the mitochondria, recording mitochondrial membrane potential (MMP). Similar to rotenone and paraquat, we found that Auf lowered the MMP Fig. S. These rNext, we wanted to assess the effects of SD and SDA on Auf- or rotenone-induced cell morphology. SH-SY5Y were incubated with 3 µM Auf for 30 min, washed, and incubated for 3.5 h at 37 °C with or without SD or SDA. As shown in Fig. SNext, to investigate the possible anti-apoptotic/anti-inflammatory activity of SD and SDA we explored Auf-induced JNK phosphorylation in SH-SY5Y cells. The cells were treated with 3 μM Auf for 30 min, washed, and then incubated for 3.5 h at 37 °C with or without SD Fig.  or SDA  see Fig, at the MAPK phosphorylation was examined in SH-SY5Y cells. The cells were treated for 30 min with 3 μM Auf, washed, and incubated for 3.5 h at 37 °C, with or without SD   Fig. S. This reMAPK phosphorylation in SH-SY5Y cells. The cells were treated for 30 min with 3 μM Auf, washed, and then incubated for 3.5 h at 37 °C, with or without increasing concentrations of LDEE .The antioxidant activity of SD and SDA was compared to another levodopa precursor, LDEE , testingEE Fig. S. As oppoµM Fig. S.MAPK phosphorylation are compared Copper ions play a role in synucleopaties, while the Cys-sulfhydryl group are known as copper chelators. These activities led us to investigate the effect of SD or SDA on rotenone-induced expression of α-syn in SH-SY5Y cells. Cells treated for 60 min with SDA Fig.  or SD F at the i12The efficacy of SDA in preventing α-syn aggregation was tested also in HEK293 overexpressing α-syn. HEK293 cells were transfected with cDNA encoding wt α-syn. Forty-eight hours after transfection, the cells exhibited high levels of monomers, and aggregated forms of α-syn, monitored by the corresponding α-syn antibodies Fig.  see Fig. PreincuSlowing the progression of PD remains a critical need in the therapy of the disease. Levodopa, the presently used treatment like dopamine itself, is known to form toxic semiquinones by auto‐oxidation. This in turn leads to a decrease in GSH level and cell death (review ).In the present study our goal is to characterize two novel levodopa precursors comprising of anti-inflammatory/anti-apoptotic activities, which may well protect DA neurons from neuroinflammation and premature cell death. Small molecular weight levodopa analogs were prepared, in which the antioxidant NAC is linked via a peptide bond to either the N-terminal of L-DOPA-Cys-amide, forming SD or to the N-terminal of L-DOPA amide, forming SDA. Both SD and SDA were designed to provide NAC and Cys into nigral DA cells concomitantly with L-DOPA delivery, aimed at replenishing the missing dopamine simultaneously with reversing oxidative stress/inflammatory damages. Treatment with SD and SDA having multifaceted activities combined into a single molecule have the potential to delay the onset of both motor fluctuations and dyskinesias and slow premature loss of nigrostriatal denervation of the dorsal putamen and nigral cells.To assess the neuroprotective properties of SD and SDA in vivo, we used the systemic acute rotenone rat model that reproducibly mimics many aspects of the pathology of human PD . In thisSince the rotenone-treated animals develop bradykinesia, postural instability, and/or rigidity , this moMotor coordination and balance were monitored in the rotenone/only, rotenone/SD and rotenone/SDA treated rats, using rotarod, beam walking, and rearing activity. Postural instability, and/or rigidity were developed by all rotenone/only-treated animals, and reversed in the rotenone/SD- or rotenone/SDA-treated rats. The significant rescue of impaired motor activities subsequent to intraperitoneal injection strongly suggests that both SD and SDA may well cross the BBB, reversing the rotenone-triggered toxicity of the DA neurons. The intraperitoneal injection of the TXM-peptide, TXM-CB3, was previously shown to reverse inflammatory consequences in the brain of diabetes Zucker rats . These rred). This ASK1/Trx1red complex is unable to activate the MAPK-apoptotic pathway. Upon oxidation of Trx1red to Trx1ox, the complex dissociates enabling free ASK1 to activate the MAPK-inflammatory/apoptotic pathway.It is well established that the inflammatory/apoptotic pathway is activated by the apoptosis signal-regulating kinase1 (ASK1) . ASK1 foThe anti-inflammatory/antioxidant properties of SD and SDA have been demonstrated in vitro, monitoring the activation of MAPK pathway in the neuronal human neuroblastoma SH-SY5Y cells. Oxidative stress was induced in the cells by Auf, an organogold selective inhibitor of thioredoxin reductase, known to activate ASK1 by maintaining Trx1 in the oxidized form. Through the activation of the MAPK-inflammatory/apoptotic pathway, Auf also inhibits the mitochondrial Trx2 causing the failure of mitochondrial function in the brain . Furtherox to Trx1red with resultant activation of ASK1, and/or a direct effect of ROS scavenging and elevation of GSH levels. These effects establish the anti-apoptotic/anti-inflammatory activities of SD and SDA in neuronal cells, promoting potential clinical relevance in protecting nigrostriatal denervation of the dorsal putamen and slowing nigral cell loss.The ability of SD and SDA to reverse the Auf-oxidizing effects implies a dual mechanism of action. SD and SDA exert an indirect antioxidant effect by reducing Trx1The in vivo studies suggest that SD or SDA, which have both NAC and levodopa moieties within them, appear to cross the BBB, thereby facilitating delivery of both components to the targeted cells. This is in contrast to NAC which is BBB-impermeable. Acting as a vehicle to bring NAC into the brain, SD and SDA might provide antioxidant protection to DA neurons, simultaneously with replenishing the missing dopamine in the nigral cells. The data suggest that the combined activities of SD and SDA into a single molecule, might have a significant advantage over NAC and levodopa administrated separately. In the absence of a clinically efficient neuroprotective treatment to slow PD progression, SD and SDA might have a putative disease-modifying effects by conferring antioxidant/anti-apoptotic activity within the DA neurons.Alpha-synuclein (α-syn) is abundantly expressed in neurons and is the major constituent of Lewy bodies, which are the hallmarks of neurodegenerative diseases called synucleinopathies, which like PD, results in multiple system atrophy, Lewy body dementia, Alzheimer’s disease, and frontotemporal dementia.The prevalence of misfolded fibrillar aggregates of α-syn associated with Lewy bodies is consistent with the role of copper ions and oxidative stress, forming highly toxic α-syn oligomers and α-syn aggregation . Copper Since sulfhydryl groups are known to chelate copper ions, SD and SDA were examine for the effects on α-syn expression and aggregation.Both SD and SDA showed a reduction in α-syn expression in SH-SY5Y cells exposed to a high dose of rotenone. Also, production of aggregated forms of α-syn was significantly lower in HEK293 cells overexpressing α-syn in the presence of SDA. The precise mechanism by which SD or SDA prevent stress-induced α-syn expression in neuronal cells is not yet clear and requires further study.The second experiment investigated a direct effect of SDA on α-syn oligomerization, using α-syn transiently transfected HEK293 cells, which express high levels of α-syn monomers and oligomers . PreincuThe newly designed levodopa precursors, SD and SDA appear to relieve motor impairment in the rotenone rat model and inhibit oxidative stress-induced activation of the inflammatory MAPK-apoptotic pathway in neuronal cells. Initial data also suggest that SDA reverses α-syn oligomerization.To date, there is no clinically disease-modifying treatment that can halt PD progression. The failure to develop neuroprotective or disease-modifying strategies results mainly because treatment starts with the appearance of motor symptoms, long after a significant nigral cell loss. Nevertheless, the standard treatment of levodopa, which is almost entirely centered on dopamine replenishment performs rather efficiently during the initial 4–5 years of treatment, implying viability of the remaining DA neurons. Therefore replacing levodopa treatment with SD or SDA may possibly attenuate the loss of the remaining nigral cells and slow PD progression. Future toxicology studies and subsequent clinical testing of SD or SDA alongside levodopa could establish their potential clinical benefits and advantage in halting disease progression.The combined redox activity and levodopa release with lowering α-syn aggregation might potentially slow-down nigral cell loss, protect nigrostriatal denervation of the dorsal putamen, and attenuate Lewy pathology. Hence, SD and SDA have the potential to become a disease-modifying treatment of PD or other LB-like pathologies.Auranofin , triethylphosphine  gold(I); LDEE from TEVA Ltd Israel. SD, SDA, and TXM-peptide TXM-CB3, and AD4 (NAC-amide), were custom synthesized by Novetide, Ltd, Haifa, Israel. All other materials were purchased from Sigma, Jerusalem. α-syn plasmid was a kind gift of Dr. R. Sharon .2, as previously reported [2 in RPMI 1640 supplemented with: l-alanyl-l-glutamine (4.4 mM); 10% fetal bovine serum; penicillin (100 U/ml) and streptomycin (100 μg/ml); HEPES pH 7.3 (10 mM). Cells were plated at a density of 6.25 × 104/cm2 and incubated for 24 h, after which they were exposed to different treatments. Tissue culture serum and medium were from Biological Industries .Human neuroblastoma SH-SY5Y cells were cultured in DMEM/F12 HAM 1:1 medium supplemented with 10% fetal bovine serum (FBS) and penicillin–streptomycin, incubated at 37 °C with 5% COreported . Human eThe anti-inflammatory activity of SD and SDA was tested essentially as previously reported for TXM-CB3 . SH-SY5YMAPK (Thr180/Tyr182), rabbit mAb; p38, rabbit Ab; GAPDH ; α-syn, Abcam, Cambridge, UK EPR20535; β Catenin, mouse mAb  diluted in 5% BSA, 0.04% Azide in TBS-T. Proteins were detected with Anti-Mouse or Anti-Rabbit IgG-HRP linked antibody .Western blot analysis were performed essentially as previously published . Twenty HEK293 cells were plated on collagen (rat tail)  and incubated for 16 h in 24-well plates and allowed to reach 70–80% confluency in 1 ml of culture medium. The next day, the cells were washed twice with regular DMEM, incubated at 37 °C, for 5 h in Opti MEM  containing the transfection mixtures of 0.75 μg plasmid cDNA encoding wt α-syn and polyethyleneimine (PEI) at a ratio of 1:3 (DNA to PEI) in 400 µl Opti MEM after vortex for 10 s, and incubated at RT for 15 min. After 5 h the medium was replaced by regular DMEM containing 10% FBS solution. Cell media was replaced after overnight incubation, with DMEM containing 10% FBS solution with or w/o 500 µM of SDA and incubated for 48 h. Then the cells were lysed with 0.12 ml ice-cold lysis buffer , 10% glycerol, 0.6% SDS, bromophenol blue, supplemented with 7 μl β-mercaptoethanol, and heated overnight at 65 °C. Proteins were separated on SDS-PAGE α-syn monomers, dimers, and trimers were detected after blotting to nitrocellulose with the corresponding α-syn antibodies.P value < 0.05; **P value < 0.01.SH-SY5Y cells were treated with increasing concentration of SD or SDA for 60 min. Then the cells were washed and incubated with 5 µM rotenone for 16 h. The level of α-synuclein in cell lysates was determined after protein separation on 12% SDS-PAGE using α-syn antibodies. The values calculated by densitometry shown as averages (±SEM) of two independent experiments normalized with housekeeping level of β-catenin; Student’s t-test (two populations) was performed for rotenone-treated cells. *A commercial company Science in Action, Nes-Ziona, Israel, uses its ethical permission to do animal studies as an outsourcing service (#C148210).2 and decapitated.All animals were treated according to the National Institute of Health (NIH) guidelines for the care and use of laboratory animals. Animal ethics committee accredits the company, and licensed veterinarians conducted the experiments. Rat were sacrificed by an overdose of COoC. Twenty male Sprague Dawley rats 7-8 weeks  were divided into 4 experimental groups. The four groups comprised of rotenone only (n = 6), rotenone with SD (33 mg/kg) (n = 6), rotenone with SDA (33 mg/kg) (n = 6), and naive rats (n = 2) that were not treated and kept under the same conditions throughout the experiment. SD and SDA were administered intraperitoneally (i.p.) once a day in the afternoon (days 1–9).The experiments were performed by “Science in Action” Rehovot, according to Cannon et al. . RotenonAnimals body weight was determined before initiation of treatment (day 0), on days 4, 7, 10 during the experiment and on termination day 11. On day 11 the animals were sacrificed.Rearing behavior, rotarod, and beam walk tests were performed before initiation of treatment (day 0) and on days 4, 7, and 10 of the experiment.Motor coordination and balance was evaluated on the rotarod, which was set to accelerate from 4 to 40 rpm in 300 sec. Animals were placed at separate lanes on the rotarod with initial rotation set on 4 rpm.Animals were placed in a clear glass cylinder (40 cm high × 20 cm diameter) and number of rears in 2 min was monitored. Rear was considered when animals raised their front legs above the shoulder and made a contact with the wall of the cylinder with their forelimb.Animals were trained for 2 days to traverse the length of the beam. On the day of the test animals were gently placed on the 1 m long narrow aluminum beam facing one of the ends and allowed to walk along the beam, monitoring the time taken to reach the end of the beam.P < 0.05 level [All values were presented as mean ± standard deviation (SD) or standard error of mean (SEM), and differences were considered to be statistically significant at the *05 level . StatistMarked-up manuscriptSupplemental Material


Players who completed more than 60 min in the previous game had significantly increased pregame CK levels and fatigue in multi-match weeks. Midfielders had both significantly increased pregame CK and muscle soreness in multi-match weeks. Midfielders and players with an exposure time of at least 60 min showed higher pregame CK values that should play a key role for deciding substitutions.The current study was conducted to compare muscle damage biomarkers in single- vs. multi-match weeks in elite soccer players for two consecutive seasons. A secondary objective was to analyze the influence of playing position and exposure time on muscle damage in single- vs. multi-match weeks. This is a prospective cohort study performed in a professional elite soccer club in the English Premier League during the 2018–2019 and 2019–2020 seasons up until the lockdown due to the COVID-19 pandemic. Data were collected in the Medical Department Room of an English Premier League Club before and after the soccer game from a total of 29 elite soccer players  who were enrolled in the club during both seasons. The main outcome measurements were creatine kinase (CK), weight, lean mass, % fat DEXA, high speed running, total distance, density of total distance and high-speed running and wellbeing questionnaires. Significance was set at  Professional sport has been affected by the COVID-19 pandemic . Since lThis extraordinary situation may have several consequences in clinical practice, provided that insufficient recovery may reduce athletic performance and increase the risk of sport-related injuries ,3. In faIn a more detailed way, creatine kinase (CK) levels have been used to monitor muscle damage in elite soccer players ,8,9,10 aThe current prospective cohort study was conducted to compare muscle damage in single- vs. multi-match weeks in Premier League soccer players for two seasons in a row. A secondary objective was to analyze the influence of playing position on muscle damage in congested versus standard schedules.This is a prospective cohort study performed in a professional elite soccer club in the English Premier League during the 2018–2019 and 2019–2020 seasons up until the lockdown due to the COVID-19 pandemic. The current study was approved by the Ethics Committee of Camilo José Cela University  and adhered to the tenets of the Declaration of Helsinki [Data were collected from 29 elite soccer players  who were enrolled in the club during both seasons. Goalkeepers were excluded due to their specific role in the team. The positional breakdown assigned to the players was: defender, midfielder, and striker ,21,22. AData were related to 38 matches in the 2018–2019 season and 29 matches in the 2019–2020 season. Since fixtures were not distributed homogeneously during the whole season, weeks were considered as multi-match weeks or single-match weeks when the time between matches was shorter or longer than 4 days (96 h), respectively . So, for−1), walking (0.2–2 m.s−1), jogging (2–4 m.s−1), running (4–5.5 m.s−1), high-speed running (5.5–7 m.s−1) and sprinting (>7 m.s−1), as in previous investigations [Match performance data were collected across both seasons by the English Premier League—with TRACAB  in 2018–2019 and Second Spectrum  in 2019–2020 installed at the stadiums of the home team. The data supplied provided different information about total distance and different velocities of the players during the game. Kick off time was from 12:30 to 19:00 depending on the fixture and television broadcasting. To develop the player’s activity profiles, movements were coded into the following categories and speed thresholds: standing  in combination with a spring-loaded AccuChek lancet device . A 30 μL capillary blood sample was placed on the measurement strip and analyzed by Reflotron Plus  system according to the manufacturer’s recommendations. Capillary blood was analyzed using this method and displayed an intraassay reliability of <3% coefficient of variation . Pre-gamWellbeing questionnaires were collected everyday through a software program  on an iPad Air  located in the changing room of the players. Players were familiarized with this questionnaire, which was distributed according to previous recommendations ,28,29 anBody fat and lean mass percentages were evaluated by means of dual-energy x-ray absorptiometry (DEXA) using a total body scanner  according to the manufacturer’s procedure. All the scanning and analyses were performed by the same specialist to ensure consistency.p < 0.05.All statistical tests were performed using the package IBM SPSS Statistics v. 26.0 . A descriptive study was carried out to analyze the data. Continuous variables were presented as mean values and standard deviation (SD) along with 95% confidence intervals (CI). When appropriate, data were provided as percentages. The Shapiro–Wilk test was used to identify normal distribution of the data. The Mann–Whitney U test was used to analyze differences between multi-match weeks and single-match weeks. One-way ANOVA or multivariate data analysis was used to test the profile of the values, depending on the playing position, in the congested weeks and non-congested weeks. Analysis was performed at the 95% confidence level. Significance was set at Out of the 55 analyzed matches, 22 were considered to belong to multi-match weeks and 33 to single-match weeks. In addition, CK pre-match multi-match data involved a mean of 9.77 players per match, while single-match data involved a mean of 9.75 players per match.p = 0.029) (p = 0.015) (p = 0.036) (p = 0.029). No more significant changes were found in this group. These results are listed in Players who completed more than 60 min in the previous game had significantly increased pregame CK levels in multi-match compared to single-match weeks  . In addi= 0.015)  and dens= 0.036)  in multip = 0.045). Similarly, midfielders’ muscle soreness  and total distance  were also significantly higher in multi-match vs. single-match weeks. Conversely, no significant changes were found in defenders and strikers. These results are summarized in Regarding player position, our results clearly demonstrate that midfielders had pregame CK values that were significantly increased in multi-match weeks when compared to single-match weeks (295.16 ± 185.26 vs. 240.58 ± 134.97; To the best of our knowledge, this was the first study that compared muscle damage in single- vs. multi-match weeks for two consecutive seasons in English Premier League soccer players.Our results clearly demonstrate that pre-game levels of CK were significantly higher in congested compared to non-congested weeks when players played more than 60 min. Similarly, internal training load expressed as fatigue and muscle soreness were also significantly increased. As the biomarker is an individual independent value, clinically this difference suggests that changes in pre-game CK levels intra-individually in a congested week would help us to understand how close the player is to the baseline of a single-match week. This pre-game CK level could be taken as an important factor by the coaches and the medical department when deciding the availability of players for the next game. Conversely, in spite of post-game CK levels being higher in multi-match weeks, differences were not statistically significant with respect to single-match weeks. The latter finding could be explained considering that in periods of congested scheduling, soccer players reduced the number of low and medium intensity actions they participated in, but maintained the number of higher intensity actions . In addiOur study also shows that there is no difference in total distance between players due to playing in a single- or multi-match week, which is similar to other previous research , and posIt is widely accepted that there are position-specific differences in soccer players’ match performance ,36,37. ARecent studies have emphasized that professional soccer matches can be carried out safely during the COVID-19 pandemic . It was The strengths of the current prospective cohort study include that it was conducted in a professional soccer club in the English Premier League for two consecutive seasons. In contrast to previous cross-sectional studies  or shortThe present study had some limitations that should be also addressed. Firstly, data from a single club were assessed, meaning that the current results may have limited application. Secondly, considering the fact that collecting data during official top-level competitions is a complex institutional mission within a particular ecological environment, we could not repeat postgame CK levels in a strict 24- or 48-h cycle after the end of the match, as shown in previous studies on this topic ,34.Thirdly, it is well-known in the related literature that CK values are individualized and highly dependent on multiple factors as lean mass , race 4, the levIt was concluded that pregame CK levels were significantly increased in multi-match weeks when players had less than 4 days of recovery between consecutive matches. Conversely, no significant changes were found in postgame CK levels, suggesting that player rotation and early recovery strategies were adequate in both single- and multi-match weeks. Regarding playing position, midfielders exhibited higher pregame CK levels when compared to forwards and defenders. Similar results were reported in soccer players who played more than 60 min in the previous game. Accordingly, individualized training loads and recovery protocols are strongly encouraged. Furthermore, prospective cohort studies, involving a larger sample of clubs from elite soccer leagues, are necessary to confirm the current findings.


Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOAu of Drosophila simulans (94.46% identity) and WOCin2USA1 of the cherry fruit fly, Rhagoletis cingulata (99.33% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40–62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species. Wolbachia, obligate intracellular bacteria belonging to the order Rickettsiales [Wolbachia cause phenotypes such as cytoplasmic incompatibility (CI) and feminization in arthropods, or support growth and reproduction in filarial nematodes [Wolbachia-induced phenotype in insect hosts and presents a form of conditional sterility whereby crosses between infected males and uninfected females produce unviable offspring; infected females may successfully mate with Wolbachia-infected or uninfected males, conferring them a selective advantage [Wolbachia are divided into several supergroups based on their ftsZ gene sequence, with supergroups A and B found exclusively in arthropods and supergroups C and D found exclusively in nematodes [Wolbachia are abundant in male and female germlines and are enriched along the reproductive tract, but also present in somatic structures  of select host species. Transmission is predominantly vertical, from female to offspring, although horizontal transmission has been documented in nature [Wolbachia (phage WO) were first discovered in 2000 and remain one of few published cases of bacteriophages that infect intracellular bacteria [Wolbachia taxa in the supergroups A and B, but are believed to be absent from Wolbachia supergroups C and D [Wolbachia or arthropod host [Wolbachia density and therefore, affect development and phenotype of its eukaryotic host [Wolbachia with accessory genes for cytoplasmic compatibility and male killing [It is estimated that 66% of all insect species and the majority of filarial parasites that infect humans are infected/colonized with ttsiales  Wolbachiematodes , 3. Cytodvantage , 5. Wolbn nature , 5, 7. Abacteria . Phages  C and D . The perpod host . Phage Wtic host . Further killing , 13.Wolbachia genomes have been sequenced and phage WO is of interest for being the only known mobile genetic element in Wolbachia, and its hypothesized role in generating the high level of diversity seen among Wolbachia today [Wolbachia strains mediated by WO phages [Wolbachia phages, sequence data is limited to the minor capsid protein-coding gene, and there remain entire families and genera of Wolbachia-harboring arthropods in which phage has not yet been described [Gryllidae) of the genus Allonemobius (ground crickets), whose members include A. socius (the southern ground cricket) and A. maculatus (the spotted ground cricket), found throughout North America. Wolbachia belonging to supergroup B has been identified in A. socius (strain: wSoc), where it is hypothesized to play a role in lengthening female crickets’ spermathecal ducts, thus increasing their control over mate choice. [Allonemobius.In recent years, an increasing number of ia today , 14. EviO phages . For a mescribed . One suc choice. –18. HoweAllonemobius crickets (phage WOSoc) and estimated its prevalence in a local A. socius population. We characterized the novel phage WOSoc by immunohistochemistry, transmission electron microscopy, and whole genome sequencing, expanding the limited set of fully described bacteriophages of Wolbachia by adding this novel bacteriophage for which we provide evidence of phage particle production, and complete genes which may mediate bacterial cell wall lysis and manipulation of host translation.In the present study we identified, for the first time, a phage WO in A. socius crickets (n = 40) were collected in the summer of 2019 from Forest Park, St. Louis, Missouri, USA . Crickets were sexed based on the presence  or absence  of an ovipositor and ecological data including morphological features and geographical distribution were used to confirm species identification. All insects were euthanized by placement at -20° C for 30 minutes before dissection and homogenization of abdomens in 500 μL of phosphate buffered-saline by 15-minute high-intensity beating with a 3.2 mm chrome Disruption Bead  on the Vortex-Genie 2 mixer . The homogenate was spun down, and DNA was prepared from the supernatant using the DNeasy Blood & Tissue Kit  according to manufacturer recommendations, with elution into 100 μL sterile water and storage at -20°C or 4°C until use.Adult Wolbachia surface protein (WSP) gene [Wolbachia phage capsid protein (WPCP) gene [Conventional PCR reactions with total cricket abdomen genomic DNA template were run using previously validated primers to the conserved SP) gene  and to tCP) gene . PCR waswSoc and WOSoc sequences  for 1 hour at room temperature or overnight at 4°C using the alkaline phosphatase-anti-alkaline-phosphatase (APAAP) technique according to the manufacturer’s protocol . Hybridoma supernatant was kindly provided by Dr. Patrick Lammie and the antibody was purified as described previously [B. malayi worms from previous studies [FAST Fast Red TR/Naphthol AS-MX (Millipore Sigma) Tablets were used, and sections were counterstained with Mayer’s hematoxylin (Millipore Sigma). Sections were analyzed using an Olympus-BX40 microscope and photographed with an Olympus DP70 camera.For immunohistology, 10 whole eviously . All ant studies  were useHigh molecular weight (HMW) DNA was purified from a homogenate of a whole single adult female cricket prepared by 15 min beating with a lead bead using the MagAttract HMW DNA Kit (Qiagen) according to manufacturer specifications, eluting in 100 μL sterile water. Presence of HMW was visualized by gel electrophoresis as a dark band  above the 15 kb DNA ladder limit . Presence of WPCP in HMW DNA was confirmed by qPCR. DNA was then purified further using AMPure XP beads  at a ratio of 1.8:1 bead to DNA sample. Library was prepared according to Oxford Nanopore’s 1D Genomic DNA Ligation Protocol (Version GDE_9063_v109_revA) using the LSK-109 Ligation Sequencing Kit  with DNA fragments of all sizes purified using the Short Fragment Buffer. 60 μL of library containing 12 μL genomic DNA was loaded as input into the flow cell and the sequencing reaction run for 20 hours using MinKNOW GUI software (Oxford Nanopore Technologies) set to the High Accuracy Flip-Flop Model, generating 6.1 giga base pairs of data (estimated N50: 2.46 kb). Basecalling of Fast5 files into Fastq format was performed using Guppy neural network basecalling software . Base stWolbachia phage WO (taxid:112596); features automatically assigned “hypothetical protein” were relabeled with known function if homologous  to a described phage protein. Genomic features were visualized in scaffolds independently and manually color-coded by function using Gene Graphics visualization application [Putative WOSoc reads were extracted by mapping MinION sequences against published phage WO reference genomes using Minimap2 software . Mapped lication .Primers were manually designed to amplify phage tail and capsid regions based on the MinION reads . ConventDNA sequences of phage WO open reading frames 2 (ORF2) and 7 (ORF7), respectively coding for the large terminase subunit and minor capsid, are biomarkers known to produce highly congruent phage WO phylogenies . NucleotA. socius males and females (N = 70) were euthanized and thoroughly homogenized in 40 mL of SM buffer . Homogenate was incubated on ice for 1 hour followed by 11,000xg centrifugation for 10 minutes at 4˚C to remove debris. Solid polyethylene glycol (PEG) was added to homogenate to a final concentration of 10% and mixed by manual shaking for 1 minute, followed by an additional 1-hour incubation on ice and 11,000xg centrifugation for 10 minutes at 4˚C. Supernatant was discarded and the remaining pellet was resuspended in 10 mL of SM buffer. To the suspension, an equal volume of chloroform was added followed by centrifugation at 3,000xg for 15 minutes at 4˚C to remove the PEG. The aqueous layer containing phage was filtered through a 0.22 μM vacuum filter to remove Wolbachia and other bacteria. Phage lysate was concentrated using Amicon Ultra-15 100 kDA Centrifugal Units  according to [Phage was purified according to the protocol described in  with slirding to  and recoA. socius, ovaries were dissected and adsorbed to an electron transparent sample support (EM) grid. Tissue was washed in PBS and fixed in 1% glutaraldehyde for 5 minutes at room temperature, followed by two 30-second washes with deionized water. Phage particles were negatively stained in 1% uric acid for 1 minute and wicked gently and placed in a grid box to dry. Phage suspension was processed identically, with 50 μL of the concentrated suspension adsorbed to an EM grid. Samples were observed on a JEOL 1200 EX transmission electron microscope  equipped with an AMT 8-megapixel digital camera From freshly caught adult female Wolbachia by TEM, one half of the ovaries of each of 6 crickets was fixed in 2% paraformaldehyde/2.5% glutaraldehyde  in 100 mM phosphate buffer, pH 7.2, for 1 hour at room temperature. The other half of the ovary sample was added to 1X PBS for DNA extraction and confirmation of Wolbachia presence by PCR. Only samples that were positive by PCR for Wolbachia were further processed for TEM. These samples were washed in phosphate buffer and post-fixed in 1% osmium tetroxide (Polysciences Inc.) for 1 hour. Samples were then rinsed extensively in distilled water prior to staining with 1% aqueous uranyl acetate  for 1 hour. Following several rinses in distilled water, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome , stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc.) equipped with an AMT 8-megapixel digital camera (Advanced Microscopy Techniques) [To confirm the presence of phage in hniques) .Wolbachia in crickets. In order to confirm the DNA sequence of WSP of Missouri crickets, DNA was amplified by conventional PCR using the pre-validated WSP primers. WSP sequence showed 100% identity to WSP of A. socius from Virginia (Accession: AY705236.1). A 400 bp amplicon of phage DNA was amplified by conventional PCR using pre-validated primers corresponding to nucleotide positions 7353–7761 of phage WO of cricket Teleogryllys taiwanemma cricket and showed close homology to the capsid protein genes from phage WO of Supella longipalpa  and Cadra cautella . The A. socius WSP and phage WOSoc WPCP gene sequences were used to design SYBR-based real-time PCR assays for WSP and WPCP, respectively. Using the strict CT cutoff of 23 cycles, we determined that from 40 insects sampled 19 (47.5%) were positive for both WPCP and WSP DNA via qPCR with our optimized primers; three samples (7.5%) were WSP-positive but WPCP-negative  as a reference genome, we identified 511 homologous WOSoc reads from the MinION run of whole-cricket homogenate HMW DNA with an average quality per read (Phred Score) of 23, corresponding to an overall base call accuracy exceeding 99%. From these reads, we assembled 12 contigs totaling 53,916 bp at an average depth of 14.6X and a GC content of 35%. After confirming and extending these contigs with Illumina reads and removing low quality reads and reads derived from the Wolbachia genome, the WOSoc genome was captured in 4 high-quality scaffolds totaling 55,288 bp , while phage WOSoc ORF7 was most similar to WOCin2USA1 of cherry fruit fly, Rhagoletis cingulata , both insects of the order Diptera.  and vector competence [Wolbachia populations is important to optimize these intervention strategies, which are limited by Wolbachia’s host specificity and phenotypic effects. Future studies are needed to show whether phage WOSoc can be utilized to manipulate Wolbachia in A. socius or other host species infected by Wolbachia.ematodes  and as aematodes . Traditiematodes , 63. Trampetence –66. A beS1 FileSequences in FASTA format of the assembled scaffolds 1–4, the head decoration protein, major capsid protein, and minor capsid protein.(DOCX)Click here for additional data file.S2 FileGenome annotation showing start and stop of 63 open reading frames.(XLS)Click here for additional data file.


Plasmodium vivax was the predominant species (96.6%). The number of indigenous cases decreased gradually and since 2017, no indigenous cases have been reported. Malaria cases were mainly distributed in the southern and southwestern areas of the county; 55.6% of the indigenous cases were reported in Nabang Township, which also had the highest risk of imported malaria. The “1–3–7” approach has been implemented effectively, with 100% of cases reported within 24 h, 88.9% cases investigated and confirmed within 3 days and 98.5% of foci responded to within 7 days. Although malaria elimination has been achieved in Yingjiang County, sustaining elimination and preventing the re-establishment of malaria require the continued strengthening of case detection, surveillance and response systems targeting the migrant population in border areas.Yingjiang County, which is on the China–Myanmar border, is the main focus for malaria elimination in China. The epidemiological characteristics of malaria in Yingjiang County were analysed in a retrospective analysis. A total of 895 malaria cases were reported in Yingjiang County between 2013 and 2019. The majority of cases occurred in males (70.7%) and individuals aged 19–59 years (77.3%).  It has been estimated that there were at least 30 million malaria cases annually before 19492. Following the implementation of a significant integrated malaria control and elimination programme for more than seven decades and specifically the “1–3–7” surveillance and response strategy that was developed and has been implemented since 20104, the disease burden has been greatly reduced5. Since 2017, no indigenous cases have been reported, achieving the standard of national malaria elimination6. Currently, China is ready for nationwide malaria elimination certification by the World Health Organization (WHO).Malaria, an infectious disease, has had the longest epidemic duration, and the broadest impact in the history of China7. According to malaria epidemiological data collected from surveillance systems in China's border areas, the population crossing the border and border residents of Yunnan Province, China, have a higher risk of malaria transmission8. Historically, malaria has been serious in Yunnan Province with more than 400 thousand malaria cases10. Since 2010, Yunnan has been the only province with Plasmodium falciparum transmission in China11. The last indigenous malaria case in China was reported in Yingjiang County of Yunnan Province in 20166; there have been no indigenous cases reported nationwide since, which allowed Yunnan Province to achieve verification of malaria elimination in June 2020.Yunnan Province borders Myanmar, Laos, and Vietnam; the border extends 4060 km, touching 25 counties, with 17 border ports, and 643 border crossings. There is no natural barrier along the border10. The border areas on both sides are rural, hard-to-reach, poverty-stricken areas inhabited by minority nationalities13. The county has nine townships bordering Myanmar, five entry-exit highways, two provincial ports, four passages, and 33 boundary passages14. The climate, landscape and vectors of malaria on both sides of the border are similar13. In the last 10 years, the incidence of malaria in Yingjiang County has declined remarkably and this progress was made possible through greater access to effective malaria control tools. The last indigenous malaria case in China was caused by P. vivax infection in April 2016, located in Taiping Township of Yingjaing County6.A total of 18 counties in Yunnan Province sit on the China–Myanmar border. One of them, Yingjiang County, is located in western Yunnan Province and having a border length of 214.6 km; it borders Kachin State, Myanmar, in the western region15. According to a malaria report from the China–Myanmar Joint Prevention and Control Information Exchange Mechanism, thousands of malaria cases were reported in Laiza in 2016 and 2017. However, the number of malaria cases has decreased dramatically in the last three years, supported by cross-border cooperation to achieve malaria elimination, timely case detection and appropriate treatment, and vector control and surveillance. The number of cases increased in 2020 as an effect of the COVID-19 pandemic . The majority of cases occurred in individuals aged 19–59 years . Most patients were outdoor workers , who have the highest risk of malaria infection (P < 0.0001); while indoor workers accounted for 27.8% (249/895) of the cases.A total of 895 malaria cases were reported in the entire county in 2013–2019, including 45 indigenous cases and 850 imported cases. All cases were diagnosed by microscopy, and except 7, were also confirmed by polymerase chain reaction (PCR). There were no malaria-associated deaths reported during this period. The number of malaria cases was the lowest, at 72, in 2013 and reached a peak of 186 in 2016; then, it decreased to 92 in 2019. Malaria cases occurred predominantly in males , with a sex ratio of 2.42:1, but the difference was not significant . P. vivax infection was the most common cause of malaria in Yingjiang County, as well as in Yunnan Province. The annual reported number of P. falciparum cases was less than 10 from 2013–2019, and no P. falciparum cases was reported in 2019. One P. malariae infection and two mixed infections involving P. falciparum and P. vivax were reported  in 2013 to 0.05% (1/185) in 2016. The last indigenous malaria case reported in 2016 in Yunnan Province, was also the last in the whole country. Since 2017, no indigenous cases have been reported. 8/72 in 2There are 15 townships in Yingjiang County and nine of which border Myanmar . A total of 261 foci were identified during the study period, and 98.5% (256/261) were investigated and responded to within 7 days.The “1–3–7” approach describes a system of activities dependent on timely diagnosis and response to malaria cases; the strategy includes strict hourly timelines to be followed. Within 24 h, any malaria case must be reported. By the end of day 3, the county’s Center for Disease Control and Prevention (CDC) must confirm and investigate the case and determine whether there is a risk of spread. By the end of day 7, the county CDC must perform measures or interventions to prevent further spread by testing residents and family members in the community where the malaria case was identified. From 2013 to 2019, there were no delays in the reporting of malaria cases, and 895 malaria cases in Yingjiang County were reported within 1 day. In terms of case investigation and confirmation, an average of 88.9% (range: 64.2–100%) of cases were investigated within 3 days, and this indicator reached 100% in 2014, 2015, and 2016  were diagnosed in a health facility of a county CDC/hospital or township hospital . For example, people engaged in frontier trade may frequently cross the border, while local farmers may go to Myanmar for logging or mining during the slack seasons in farming. In November 2019, the Hongbenghe Water Conservancy Construction Site, which was located at the China–Myanmar border in Taiping Township of Yingjiang County, was identified as an epidemic focus, and a total of 22 P. vivax malaria cases were identified. According to the case investigation, the source of infection was the bite of an Anopheles mosquito carrying malaria parasites that was transported from overseas; therefore, these cases were classified as imported malaria cases. This epidemic cluster caused another peak in monthly malaria cases in Yingjiang in 2019 , Yingjiang County was a Type I county  that was targeted for the implementation of key strategies to strengthen surveillance and reduce the incidence in the pre-elimination stage010–2020,P. vivax is the most geographically widespread parasite causing human malaria, and over 2.5 billion people are at risk of infection26. P. vivax malaria is mainly endemic in Southeast Asia and Central and South America. As reported by the WHO, more than half of P. vivax infections (53%) occur in the WHO South-East Asia region27. The proportion of vivax malaria cases in Yingjiang County increased from 2013 to 2019 and no falciparum malaria cases were reported in 2019, indicating that P. vivax was the predominant species on the Myanmar side and the rate of P. falciparum infection in Myanmar was very low because more than 95% of the imported cases were from Myanmar. This change was similar to that in other countries in the Greater Mekong sub-region29. The epidemiology of vivax malaria in this region is highly heterogeneous and P. vivax has become a major challenge to malaria elimination in this region31.3. In this study, we analysed the three indicators included in the “1–3–7” approach in Yingjiang County. All malaria cases were reported to the CIDRS within 24 h, indicating that the web-based case reporting system is sensitive, as it allowed a prompt response, even in township hospitals. The proportion of case investigations and confirmations completed within 3 days was 88.9%, compared with only 64.2% in 2017  pandemic has affected the availability of key malaria control interventions and may cause malaria-associated morbidity and mortality to increase in the future41.Although great achievements have been made in reducing the overall number of cases of malaria and eliminating indigenous cases in China, the elimination campaign faces challenges in areas near international bordersSubstantial gains have been made in reducing the number of malaria cases and eliminating indigenous malaria in Yingjiang County. Sustaining elimination and preventing the re-establishment of malaria require the strengthening of case detection, surveillance and response system targeting migrant populations in border areas.. The land area of Yingjiang County is 4429 km2, with a local population of 316,990 people. Yingjiang County is mountainous with several alluvial plains. The county has various climate types, ranging from tropical to subtropical and temperate zones, and has an average annual temperature of 22.7 °C and annual rainfall of 2.65 m. Intact forests exist in the mountains above 2000 m. The elevation varies from 210 to 3404 m. The county is within a very active seismic zone and was affected by violent earthquakes in 2008, 2009 and 2011. Migration, plantation and logging activities are frequent along the border42. An. sinensis and An. minimus are reported to be the dominant vectors for malaria parasites43.Yingjiang County is located in western Yunnan Province . It shares a long border with Kachin State, Myanmar and had the highest risk of malaria transmission in Yunnan Province , or PCR, or clinically diagnosed malaria, which referred to patients with malaria–like signs and symptoms but without the detection of parasites on blood examination46. Malaria case data from 15 townships were extracted to map the malaria distribution at the township level. In addition, according to the Technical Program for Malaria Elimination in China47, indigenous malaria was defined as malaria infection obtained from within the province in which the diagnosis was made, while imported malaria was defined as malaria whose origin can be traced to a transmission area outside the province in which the malaria diagnosis of malaria was made; moreover, the following criteria for imported malaria was also applied: the patient has received a malaria diagnosis; the patient had a travel history to a malaria-endemic area outside China during the malaria transmission season; and the onset time for malaria was less than 1 month after returning to China during the local transmission season.Malaria case data and demographic information, including sex, age, occupation, residential address, type of disease, date of onset, and date of confirmation were collected from the web-based Chinese Infectious Disease Report System (CIDRS) from 2013 to 2019www.R-project.org)48 and SAS software  were used for data processing and statistical analysis. The chi-squared test was used to evaluate differences among the different sub-groups; Fisher's exact test was used if 25% of the cells had expected counts less than 5. Maps were created using ArcGIS 10.1 . The indicators used to evaluate the implementation of the “1–3–7” approach were determined from the individual case reports downloaded from the CIDRS. A P value < 0.05 was considered statistically significant.R software (Version 4.0.3; R Core Team, Vienna, Austria, 2020; This study has been approved by the Ethical Review Committee of National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, No. 2019008.Supplementary Information 1.Supplementary Information 2.


Peripheral inflammatory conditions, including those localized to the gastrointestinal tract, are highly comorbid with psychiatric disorders such as anxiety and depression. These behavioral symptoms are poorly managed by conventional treatments for inflammatory diseases and contribute to quality of life impairments. Peripheral inflammation is associated with sustained elevations in circulating glucocorticoid hormones, which can modulate central processes, including those involved in the regulation of emotional behavior. The endocannabinoid (eCB) system is exquisitely sensitive to these hormonal changes and is a significant regulator of emotional behavior. The impact of peripheral inflammation on central eCB function, and whether this is related to the development of these behavioral comorbidities remains to be determined. To examine this, we employed the trinitrobenzene sulfonic acid-induced model of colonic inflammation (colitis) in adult, male, Sprague Dawley rats to produce sustained peripheral inflammation. Colitis produced increases in behavioral measures of anxiety and elevations in circulating corticosterone. These alterations were accompanied by elevated hydrolytic activity of the enzyme fatty acid amide hydrolase (FAAH), which hydrolyzes the eCB anandamide (AEA), throughout multiple corticolimbic brain regions. This elevation of FAAH activity was associated with broad reductions in the content of AEA, whose decline was driven by central corticotropin releasing factor type 1 receptor signaling. Colitis-induced anxiety was reversed following acute central inhibition of FAAH, suggesting that the reductions in AEA produced by colitis contributed to the generation of anxiety. These data provide a novel perspective for the pharmacological management of psychiatric comorbidities of chronic inflammatory conditions through modulation of eCB signaling. In peripheral inflammatory conditions, such as inflammatory bowel diseases (IBD), comorbid anxiety and depression are associated with increased disease activity, greater rate of relapse and reduced responsiveness to therapies –5, signiPeripheral inflammation, particularly within the gut, is known to be a potent activator of the hypothalamic-pituitary-adrenal (HPA) axis , 38. SusConstitutive eCB signaling constrains anxiety, as acute pharmacological disruption of eCB function rapidly produces a state of anxiety –46. SimiTo further examine the relationship between eCBs and peripheral inflammation, we utilized a rat model of colitis to investigate the potential role that the eCB system plays in the mechanisms underlying psychiatric comorbidity in chronic inflammatory diseases. Colitis represents an ideal condition for this investigation, as humans afflicted with colitis exhibit considerable psychiatric comorbidities, particularly anxiety –5, and aad libitum access to food and water. All experiments were conducted during the light phase of the cycle. All animal protocols were approved by the University of Calgary Animal Care Committee and followed guidelines from the Canadian Council for Animal Care. For each set of experiments described below, animals from a minimum of 2, and up to 4, cohorts were used, aside from locomotor activity which was assessed in a single cohort.All experiments utilized adult (~300–350 g at time of colitis induction), male or female, Sprague Dawley rats from Charles River . Animals were allowed to acclimate for at least one week prior to experiment onset. Rats were paired-housed under specified pathogen free conditions on a 12:12 h light/dark cycle with Under brief isoflurane anesthesia, rats received an intracolonic bolus of 2,4,6-trinitrobenzenesulfonic acid (TNBS) , via a cannula, inserted 7 cm proximal to the anus –77. TNBSAmbulatory activity was assessed using the Opto-Varimex-5 Auto Track  infrared beam activity monitor with a 17.5”x17.5” arena as previously described . Day 0 tAnimals were subjected to handling and body weight measurement in the behavior testing room at least 5 days prior to anxiety testing. EPM  testing occurred on Day 7 following colitis induction under dim light and with a white noise background. EPM was performed for 5 min as previously described  and is dAfter behavioral experiments were completed on Day 7 after the induction of colitis, trunk blood was collected as previously described , 84 and Excisions of brain structures were performed on ice as described previously  and sampBrain structures were excised on ice  and samp3H]-AEA to [3H]-ethanolamine [3H]-2-oleoylglycerol (2-OG) to [3H]-glycerol ) or a FAAH inhibitor (PF-04457845 (PF); Pfizer, New York, NY, USA; 100 ng and 1 µg) [As global FAAH inhibition is associated with suppression of colonic inflammation –97, we and 1 µg) –100. Inf−1, subcutaneously)) treatment, the unilateral cannula was placed into the lateral ventricle, −0.90 mm anteroposterior and 1.4 mm mediolateral from Bregma, and the pump was placed subcutaneously. Surgeries were performed on the same day, but prior to, TNBS or saline administration. One week following surgery and colitis onset, brain regions were isolated as described above for eCB analysis. Ventricular cannula placement was confirmed post-mortem with dye infusion.To understand the role of CRF signaling on the colitis-induced reductions of AEA signaling, we examined the impact of sustained disruption of CRF-R1 signaling during the entire duration of colitis  utilizing continuous drug infusion with an osmotic mini-pump  connected to a 5 mm cannula  . The osmt tests were used . For comparison of repeated measures, a repeated measure analysis of variance (ANOVA) or mixed-effect analysis was performed. For comparisons between two independent variables, two-way ANOVAs were performed. For all ANOVA analyses, significant interactions and main effects were reported, and specific group comparisons were made using Fisher’s Least Significant Difference tests. Planned comparisons based on a priori hypothesis were performed using independent t tests. t- or F-values, p values and eta squared (R2) are reported, as well as Pearson correlation coefficients (r) . Data are presented as mean ± standard error of the mean (SEM). Outliers were removed using the ROUT method [p < 0.05 was considered statistically significant. Detailed statistics for data represented in figures, as well as correlation values, are reported in Tables All statistics were carried out using Prism v8. For comparison of two groups, one-tailed  or two-tailed Student’s T method , set to T method . p < 0.0Data presented on colitis phenotype , in the amygdala and medial prefrontal cortex, but not in the hypothalamus or hippocampus   Table  in the amax; Fig. m  acutely at two doses and examined anxiety-like behavior in the EPM in order to investigate the relevance of changes in eCB levels to the increase in anxiety-like behavior.Open arm time was reduced with TNBS-induced colitis and increased with administration of the FAAH inhibitor Fig. . Based oMacroscopic damage of the colon was increased in TNBS-treated rats, but this was lower in the 1 µg PF dose compared to its vehicle Fig. . SpecifiAs increased FAAH activity was found to contribute to the generation of colitis-induced anxiety, and given previous work that showed that activation of CRF-R1 can induce FAAH hydrolysis of AEA during psychological stress , 106, weWe investigated if blocking central CRF-R1 with an antagonist, antalarmin, for the 7-days post-TNBS administration altered colitis-induced changes in AEA levels. While this caused no changes to TNBS-induced increases in macroscopic tissue damage Table , antalarp > 0.05)  in 2-AG levels in any of these areas  Fig.  were noteas Fig. . Unlike We demonstrate that the TNBS-model of colitis in rats, consistent with other rodent models of colitis , 107–109Endocannabinoid signaling is well established to regulate affective behavioral processes such as anxiety through actions localized within the amygdala, medial prefrontal cortex and hippocampus , 110. ThRecent work from our group shows that both male and female mice exhibit anxiety-like behavior in a dextran sulfate sodium model of colitis . Here weCrh [Crh expression in rodent models of gut inflammation [The finding that CRF-R1 activity mediates the colitis-induced reduction in AEA content broadens previous work indicating that CRF and FAAH exhibit an intricate relationship in the regulation of affective behavior , 106. ChCrh , 106. InCrh , 115. CoCrh , 117 shoammation , 69, 89.ammation , this efAn unexpected finding of this study was that acute central inhibition of FAAH reduced the severity of colitis. Endocannabinoids are well-established anti-inflammatory molecules , 119, anIn addition to reduced AEA, we also found that colitis was associated with elevations in 2-AG throughout several corticolimbic structures ; Alberta Innovates Health Solutions (AIHS) CRIO Project 201200828-QJP, KAS. HAV received stipend funding from CIHR (Vanier CGS), University of Calgary , AIHS and Branch Out Neurological Foundation (BONF); MM received fellowship support from AIHS and CIHR; VC received a studentship from UofC; KT received studentships from the National Sciences and Engineering Research Council (NSERC) and AIHS; KL received a studentship from BONF; AS received salary support from the UofC Mathison Centre for Mental Health Research and Education. MNH is the recipient of a Tier II Canada Research Chair. KAS holds the Crohn’s and Colitis Canada Chair in IBD Research at the UofC. Funding agencies had no influence on the design, execution or publishing of this work. MNH is a scientific advisor for Sophren Therapeutics and Lundbeck. All other authors have no disclosures to report.SupplementalSupplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Table 4Supplemental Figure 1


This editorial summarizes the contributions to the Frontiers Research topic “Innovative Analysis Ecosystems for HEP Data”, established under the Big Data and AI in High Energy Physics section and appearing under the Frontiers in Big Data and Frontiers in Artificial Intelligence journals. High energy physics (HEP) experiments are collecting an unprecedented amount of data. Major upgrades of the current HEP experiments already in progress will further increase the volume and complexity of these data. This new territory of extremely large data is inspiring and challenging physicists to devise new and more advanced analysis techniques, which feature increasingly more elaborate procedures such as machine learning and deep neural networks, to maximize the physics outcome of these studies. These developments, which will have a growing impact on HEP in the coming years, require fast and efficient analysis ecosystems and state-of-the-art hardware infrastructures. Exploiting recent advancements in data science and machine learning has already led to a significant leap in HEP data analysis. It is therefore of high interest to continue in this effect to maximize the impact of these new techniques in HEP.This research topic aimed to explore recent developments in this area with a focus on 1) innovative, user-friendly and preservable analysis frameworks and dedicated languages describing analysis algorithms, 2) fast analyses on high performance computing centers (HPCs) using modern parallelization techniques, and 3) development of analysis infrastructures based on optimization pipelines and new data formats, which can be efficiently interfaced to machine learning utilities.Šimko et al. explored a novel approach for experimental HEP data analyses based on a declarative rather than imperative paradigm to describe the analysis computational workflow. In this study, the analysis process is structured in the form of a Directed Acyclic Graph, with graph vertices representing a computation unit with its inputs and outputs, and graph edges describing the interconnections of different computational steps. The REANA analysis platform featured here allows one to express the computational analysis steps based on the declarative paradigm implemented via the Yadage workflow specification language. REANA parses the analysis workflow and dispatches its computational steps to various supported computing backends . The approach was tested on two analysis examples from the LHC: 1) reinterpretation of a search for dark matter production in association with a Higgs boson decaying to One study, Mosciatti et al., focused on improving the effectiveness of software containers which provide all dependencies needed to run a software workload independently on a host environment, enabling full reproducibility of the workloads and consequently their analysis content and results. Containers are extremely important at the LHC where software environments and operating systems become quickly outdated. One issue is that, the increase in the complexity of analysis software increases the size of container hosting the analysis, making its download and execution unpractical and slow. The study explored a new method to speed up the container download and execution by combining the so-called lazy pulling method, which allows to download image data as needed, and cache on demand in the CERN Virtual Machine File system (CVMFS). This method, which is implemented in the Kubernetes platform, reduces the container start time by at least a factor two compared to the standard download utilities.A second study Unel et al., focused on techniques to effectively express the physics content of HEP data analyses. It features a domain-specific, declarative language called Analysis Description Language (ADL) that describes the contents of an analysis in a standard and unambiguous way, independent of any computing framework. ADL can be used by experimental and phenomenological communities to facilitate the abstraction, design, visualization, validation, combination, reproduction, interpretation, overall communication and long-term preservation of analysis contents. In ADL, analyses are written in plain text files consisting of blocks separating object, variable and event selection definitions. Blocks have a keyword-expression structure, where keywords specify analysis concepts and operations. Syntax includes mathematical and logical operations, comparison and optimisation operators, reducers, four-vector algebra and commonly used functions. Analyses written in ADL can be executed on event data either via converting ADL to a compilable code by a transpiler, or via direct runtime interpretation. This study described recent advancements in ADL and in particular CutLang, a runtime interpreter of ADL.Another study Diblen et al., presents works on building a versatile vizualization tool of scientific data that allows analysis of data over the web. The study features SPOT, a tool that can be interfaced to scientific repositories such as Zenodo and HEPData. The main aim of the tools presented here is the quick and convenient re-analysis and comparisons of phenomenological data, which may lead to finding new correlations in published data. SPOT can create 1-3 dimensional visualisations by creating histograms of high-dimensional datasets, and perform interpolation to generalize data. The functionality was demonstrated and benchmarked using different examples that range from the visualization of the parameters of a 19-dimensional supersymmetry model dataset versus their exclusion status by the ATLAS experiment or their consistency with the galactic center excess model, and visualization of simulated LHC data and of the Fermi FL8Y Point Source Catalogue.Another study Adams, presented a new technique to remove background particles in events collected by the SBND detector, the near detector in the Fermilab Short-Baseline Neutrino Program, by applying deep learning on full detector images. The paper explored techniques to discriminate the origin of recorded activity to be from cosmic particles, neutrino interactions or background-noise at a single pixel level using deep convolutional neural networks on simulated data from the SBND LArTPC detector. Various DNNs were designed and different metrics were considered to optimize the prediction accuracy. The technique developed is applicable to other LArTPC detectors running at surface level, such as MicroBooNE, ICARUS and ProtoDUNE.Machine learning methods are becoming more central in HEP data analysis due to their extensive discrimination and regression powers. Another study


Background: The aim of the study was to perform a functional and structural evaluation of the anterior visual pathway in patients with Graves’ Orbitopathy (GO) using electrophysiological tests and OCT, as well as to identify potential parameters that could be useful in detecting early optic nerve damage. Methods: 47 GO patients were enrolled in the study and divided into three groups, depending on their disease severity: Group 1 with mild GO, Group 2 with moderate-to-severe GO, and Group 3 with dysthyroid optic neuropathy (DON). Pattern visual evoked potential (PVEP), flash visual evoked potential (fVEP), pattern electroretinogram (pERG), and optical coherence tomography (OCT) findings were compared between the groups. Results: In the DON Group (Group 3), N75, P100, and P2 latencies were significantly extended, whereas P100, P50, and N95 amplitudes were significantly reduced as compared to the non-DON group (Groups 1 and 2). Group 3 also had significantly thinner peripapillary retinal nerve fiber layer (RNFL) and macular ganglion cell complex (GCC). In Group 2, as compared to Group 1, P100 amplitudes were significantly reduced for all check sizes, while P100 latency was elongated for the check size of 0.9°. Group 2 also had a significantly thinner average GCC and GCC in the superior quadrant. Conclusions: Electrophysiological examinations may be of use in diagnosis of DON. OCT findings and electrophysiological responses vary in patients with different GO severity. Including regular electrophysiological evaluation and OCT in the examination of patients with GO could be of benefit. However, more research is needed to establish the true significance of pVEP, fVEP, pERG, and OCT in monitoring patients with GO. Graves’ Orbitopathy (GO) is the most common extrathyroidal expression of Graves’ disease (GD). It is an autoimmune, antibody-mediated disorder leading to the inflammation and remodeling of orbital tissues. Excessive production of glycosaminoglycans (GAGs), adipogenesis, oedema, and inflammatory infiltration induce muscle enlargement and orbital fat expansion within the constrained space of the orbit. This process may result in apical crowding and in the direct compression of the optic nerve or its blood supply .Dysthyroid optic neuropathy (DON) is a rare condition with an insidious onset, affecting approximately 5–8% of patients with Graves’ Orbitopathy. It requires urgent management as it can potentially lead to irreversible visual loss ,3. A thoThe objective of our research was to perform a functional and morphological examination of the visual pathway in patients with Graves’ Orbitopathy using pVEP, fVEP, pERG, and OCT, as well as to compare the results in patients with different degrees of severity of the disease.47 patients with Graves’ Orbitopathy were enrolled in the study, including 13 men (28%) and 34 women (72%). The patients’ mean age was 52.1 years ± 14.8 SD. The research protocol was approved by the Jagiellonian University Bioethical Committee  and the study adhered to the tenets of the Declaration of Helsinki. All patients signed a written informed consent. The exclusion criteria were other ocular diseases, high myopia greater than—6.0 D, intraocular pressure above 21 mmHg, neurological diseases , diabetes with polyneuropathy or diabetic retinopathy, and other diseases that could have an impact on electrophysiological or OCT examinations. The ophthalmological examination included visual acuity assessment, color vision assessment using Ishihara tables, tonometry, biomicroscopic anterior segment examination, cover test and Hess’s screen, indirect fundus examination, Hertel exophthalmometry, and manual kinetic Goldmann perimetry. GO severity was evaluated according to the European Group on Graves’ Orbitopathy (EUGOGO) classification  [17]. PaOptical coherence tomography was performed after pupil dilation. An RTVue OCT device  was used. An RNFL scan of 3.45 diameters, centered on the optic nerve head, was performed. The analysis of the peripapillary retinal nerve fiber layer thickness was presented in µm as Average RNFL, Superior RNFL, and Inferior RNFL.The greatest number of ganglion cells is found in the central 8 degrees of the retina; therefore, a macular ganglion cell complex (GCC) analysis was performed. The GCC consists of 3 layers of the retina, i.e., the retinal nerve fiber layer, the retinal ganglion cell layer, and the inner plexiform layer. The GCC protocol comprised 15 vertical scans and 1 horizontal scan covering an area of 7 by 7 mm, localized 1mm temporally from the macula. The analysis of GCC thickness was presented in µm as Average GCC, Superior GCC, and Inferior GCC. Additionally, the Focal Loss Volume (FLV) and Global Loss Volume (GLV) were analyzed.The patients were divided according to the EUGOGO severity classification: into mild (Group 1), moderate-to-severe (Group 2), and sight-threatening (Group 3). The groups comprised 16, 23, and 8 patients, respectively. We compared the results of electrophysiological examinations and OCT of patients with no clinical evidence of DON (Groups 1 and 2) with patients with confirmed DON (Group 3). Subsequently, we compared patients with mild GO (Group 1) with patients with moderate-to-severe GO (Group 2) in terms of their electrophysiological responses and OCT parameters.t-test and Mann–Whitney’s u-test (for independent samples).A Student’s t-test for independent samples was used to test differences between groups of patients. To test if the variances of two populations are equal, an F-test for equality of two variances was used additionally. Age and sex differences of patients between the groups were estimated using Student’s p = 0.2402 and p = 0.0771, respectively)The non-DON Group (Groups 1 and 2) comprised of 39 patients (9 men and 30 women). The mean age of patients in this group was 50.4 ± 14.7. Group 3 comprised 8 patients (4 men and 4 women). The mean age of patients in this group was 60.5 ± 12.8. The differences between the groups in terms of patients’ sex and age were statistically insignificant  with GO patients with no clinical evidence of DON (Groups 1 and 2). The comparison revealed a significant increase in N75 and P100 latencies for all check sizes and a significant reduction of P100 amplitudes for all check sizes except for check size of 1.5° in Group 3 . The difp = 0.0001) in the DON group (Group 3) and in the non-DON Group (Groups 1 and 2), respectively. There was no statistically significant difference in the P2 amplitude between the two groups (10.1 ± 7.0 vs. 12.0 ± 6.5 p = 0.2828).As for fVEP, the mean latency of P2 was 133.4 ± 16 ms vs. 118.0 ± 13.7 ms . The mean age of patients in this group was 47.1 ± 16.2.p = 0.0420), which was to be expected since men tend to have more severe GO. The percentage of men in the groups in this study increased along with GO severity. The age differences between the groups were statistically insignificant (p = 0.2490).Group 2 comprised of 23 patients (8 men and 15 women). The mean age of patients in this group was 52.7 ± 13.5. There were more men in Group 2 . Our results are in contradiction to those of Tsaloumas et al., who reported no difference in P2 latency between the DON and thyroid-associated orbitopathy (TAO) groups (112.0 ± 4.46 ms vs. 110.1 ± 2.65 ms) but found a significantly smaller P2 amplitude in DON patients (6.83 ± 0.92 ms vs. 12.4 ± 1.05 µV). Tsaloumas et al. admitted, however, that in their study, reductions in amplitude occurred more frequently than delays as compared to other studies [A visual evoked potential is produced by activated neurons of the occipital cortex. It is generated in response to visual stimulation: a flashing light (fVEP) or an alternating checkboard pattern (pVEP). The fVEP consists of several positive and negative components, among which P2 is the most commonly used in clinical assessment. We found a significantly prolonged P2 latency in patients with dysthyroid optic neuropathy, as compared to GO patients without evident signs of DON (133.4 ± 16 ms vs. 118.0 ± 13.7 ms), but we did not find any differences in P2 amplitudes between the groups  has an impact on pVEP responses . Some auThe pattern visual evoked potential is not specific to optic nerve damage as it depends on good macular function. Pattern electroretinogram is a complementary technique to visual evoked potentials. It enables simultaneous assessment of retinal ganglion cell and macular function. The P50 component of the pERG is generated by inner- and outer-retinal neurons, whereas the N95 component represents retinal ganglion cell function ,30. To tWe found significantly reduced P50 and N95 amplitudes in patients with DON as compared to GO patients without confirmed DON. The P50 latency was significantly prolonged, whereas the N95 latency was reduced in those patients. The differences in latencies, however, were much less pronounced than those in amplitudes  and wereIn patients with DON, a significant reduction in P50 amplitude may be secondary to retrograde damage of the retinal ganglion cells or may be due to ischemic changes in the outer retinal layers. The elongated P50 latency, which is characteristic of macular involvement, is more suggestive of the latter ,36. We cOur analysis of OCT parameters in GO patients revealed that patients with DON had a significantly thinner average peripapillary RNFL, as well as RNFL in the upper and lower quadrants. Our results are in agreement with the study by Park et al., who reported a significantly smaller mean temporal peripapillary RNFL thickness in patients with long-lasting DON (≥6 months) in comparison to healthy controls and patients with acute DON . In contWhen comparing the macular ganglion cell complex in DON patients and in GO patients without any clinical signs of DON, we found significant differences in all the parameters. The average GCC, as well as inferior and superior quadrant GCC, were significantly thinner in DON patients. Both the foveal loss volume (FLV) and the global loss volume (GLV) were significantly increased in DON patients. Our results are in line with the study by Romano et al., who compared patients with optic nerve compression due to GO with normal controls and found a significantly thinner average GCC, inferior GCC, as well as significantly thinner RNFL in the superior and inferior quadrant in the first group . Since tOrbital MRI was not performed in each patient, which is a limitation of this study as such scans would have revealed the factors which contributed to the changes found in the electrophysiological tests. Moreover, an additional comparison with a healthy control group would have given us a more complete picture of the morphological and functional changes in patients with GO.Dysthyroid optic neuropathy may have an insidious onset. Hence, assessing optic nerve function in electrophysiological examinations may be of use in the diagnosis of DON. In our study, we observed significantly reduced P100, P50, and N95 amplitudes, as well as significant increased N75, P100, and P2 latencies in patients with DON. Combining pVEP with pERG may be helpful in distinguishing between changes resulting from macular and ganglion cell dysfunction. fVEP may be useful in patients with severe eye surface disorders and corneal or lens opacities. Previous studies have shown that changes in electrophysiological and OCT results are likely to be present in GO patients without clinical signs of DON. In this study, patients with moderate-to-severe GO had significantly reduced P100 amplitudes, a significantly longer P100 latency for the check size of 0.9°, and reduced average and superior GCC thickness as compared to patients with mild GO. Therefore, we conclude that electrophysiological responses and OCT parameters vary in patients with different GO severity. Including regular electrophysiological evaluation and OCT in the examination of patients with GO could be of benefit. However, more research is needed to establish the true significance of pVEP, fVEP, pERG, and OCT in monitoring patients with GO.